Method for regulating and controlling ginseng adventitious root morphology and saponin accumulation

Abstract

The embodiment of the invention provides a method for regulating and controlling the form of ginseng adventitious roots and saponin accumulation. The method comprises the following steps: (a) carrying out mutagenesis treatment on initial ginseng adventitious roots by using ethyl methane sulfonate (EMS); (b) performing multiple rounds of screening and subculture to obtain to-be-selected ginseng adventitious roots with different forms and saponin accumulation; and (c) determining one or more target genes for regulating and controlling the form of the ginseng adventitious root and saponin accumulation based on the ginseng adventitious root to be selected.

Description

Technical Field

[0001] The present invention relates to the field of plant tissue culture technology, and in particular to a method for regulating the morphology of ginseng adventitious roots and saponin accumulation. Background Art

[0002] Ginseng (Panax ginseng CAMeyer) is an important medicinal and health plant. Regulation of ginseng adventitious root morphology can increase the biomass of medicinal plants and optimize active ingredients. The development of ginseng roots is regulated by a variety of biological and non-biological factors, such as light, temperature, soil type, and plant hormones. At present, the morphological regulation of ginseng adventitious roots mainly relies on rough adjustments to environmental conditions, resulting in unstable ginsenoside content and fluctuations in medicinal value. Changes in ginseng adventitious root morphology are closely related to physiological functions and the accumulation of active ingredients.

[0003] The medicinal value of ginseng is mainly due to its rich saponin components (such as Rg1, Rb1, Re, etc.), which have many significant pharmacological effects, such as anti-fatigue, antioxidant, anti-inflammatory, and immunity enhancement.

[0004] Therefore, in order to improve the medicinal value of ginseng and meet the market's growing demand for high-quality ginseng products, it is necessary to provide a method for regulating the morphology and saponin accumulation of ginseng adventitious roots to increase the accumulation of saponins in ginseng adventitious roots. Summary of the invention

[0005] One or more embodiments of the present specification provide a method for regulating the morphology and saponin accumulation of ginseng adventitious roots, the method comprising: (a) treating initial ginseng adventitious roots with ethyl methanesulfonate (EMS) for mutagenesis; (b) obtaining candidate ginseng adventitious roots with different morphologies and saponin accumulations through multiple rounds of screening and subculture; (c) determining one or more target genes that regulate the morphology and saponin accumulation of ginseng adventitious roots based on the candidate ginseng adventitious roots.

[0006] In some embodiments, the subculturing comprises 15-25 consecutive subculturings.

[0007] In some embodiments, the EMS mutagenesis treatment includes the following steps: S1: taking sterile ginseng adventitious root segments from the initial ginseng adventitious roots; S2: preparing 3 / 4MS culture medium, adding indole-3-butyric acid (IBA) and sucrose, sterilizing, and cooling for use; S3: inoculating the sterilized ginseng adventitious root segments into the 3 / 4MS culture medium, and culturing in the dark at a constant temperature; S4: preparing EMS solution, soaking the ginseng adventitious root segments after dark culture for 2 weeks in the EMS solution for the EMS mutagenesis treatment, and rinsing; S5: cutting the ginseng adventitious roots after the EMS mutagenesis treatment into small segments, inoculating them into new 3 / 4MS culture medium and culturing them for 4 weeks.

[0008] In some embodiments, in step S2, the pH value of the 3 / 4MS culture medium is 5.5-6, the concentration of the IBA is 2 mg / L-6 mg / L, and the amount of sucrose added is 1%-5%.

[0009] In some embodiments, in step S3, the constant temperature dark culture temperature is 23°C-27°C.

[0010] In some embodiments, in step S4, the concentration of the EMS solution is 0.1%-0.3%, and the soaking time is 50-70 minutes.

[0011] In some embodiments, determining one or more target genes that regulate the morphology and saponin accumulation of ginseng adventitious roots based on the candidate ginseng adventitious roots includes: extracting total RNA from the candidate ginseng adventitious roots and performing gene expression detection.

[0012] In some embodiments, the gene expression detection is to detect the expression level of the target gene in the candidate ginseng adventitious roots using real-time fluorescence quantitative qPCR technology.

[0013] In some embodiments, the target genes include PgARF1, PgIAA02, PgCYCD3, PgEXPA1, PgWOX5, PgPIN3, PgLBD16, PgCYP450, PgSUT1, PgGASA4, PgHMGR1, PgFPS1, PgSS1, PgCYP716A47, PgUGT71A27, PgSE, PgDDS, PgCYP716A53v2, PgUGT74AE2 and Pgβ-AS.

[0014] In some embodiments, the method further comprises: regulating the morphology and saponin accumulation of the target ginseng adventitious roots by regulating the expression level of the one or more target genes.

[0015] In some embodiments, the expression level of the one or more target genes is regulated by exogenous hormones, environmental condition adjustment or gene editing technology.

[0016] In some embodiments, the saponin monomer content in the selected ginseng adventitious roots is 0.01%-5%, and the total saponin content is 1%-8%; the saponin monomers are Rg1, Re, Rg2, Rg group, Rb1, Rc, Rb2, Rd, and Rb group.

[0017] In some embodiments, the target gene combination includes one or more of PgARF1, PgIAA02, PgCYCD3, PgEXPA1, PgWOX5, PgPIN3, PgLBD16, PgCYP450, PgSUT1, PgGASA4, PgHMGR1, PgFPS1, PgSS1, PgCYP716A47, PgUGT71A27, PgSE, PgDDS, PgCYP716A53v2, PgUGT74AE2 and Pgβ-AS.

[0018] In some embodiments, the target gene combination includes PgARF1, PgIAA02, PgCYCD3, PgEXPA1, PgWOX5, PgPIN3, PgLBD16, PgCYP450, PgSUT1, PgGASA4, PgHMGR1, PgFPS1, PgSS1, PgCYP716A47, PgUGT71A27, PgSE, PgDDS, PgCYP716A53v2, PgUGT74AE2 and Pgβ-AS.

[0019] In some embodiments, the target gene combination includes at least one of PgARF1, PgWOX5, PgLBD16, PgHMGR1 and PgSS1. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present specification will be further illustrated by means of exemplary embodiments, which will be described in detail through the accompanying drawings.

[0021] Figure 1 is a schematic diagram of 5 forms of ginseng adventitious roots obtained by screening as shown in some embodiments of this specification;

[0022] Figure 2 It is a schematic diagram of the expression changes of genes related to ginseng adventitious root development in different forms of ginseng adventitious roots according to some embodiments of this specification;

[0023] Figure 3 It is a schematic diagram of the expression changes of genes encoding key enzymes for ginsenoside synthesis in adventitious roots of ginseng with different forms according to some embodiments of this specification;

[0024] Figure 4 This is a schematic diagram of the differences in saponin contents of ginseng adventitious roots of different forms after culture according to some embodiments of this specification. DETAILED DESCRIPTION

[0025] In order to more clearly illustrate the technical solutions of the embodiments of this specification, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some examples or embodiments of this specification. For ordinary technicians in this field, without paying creative work, this specification can also be applied to other similar scenarios based on these drawings.

[0026] One or more embodiments of the present specification provide a method for regulating the morphology and saponin accumulation of ginseng adventitious roots, the method comprising: (a) treating the initial ginseng adventitious roots with ethyl methanesulfonate (EMS); (b) obtaining candidate ginseng adventitious roots with different morphologies and saponin accumulation through multiple rounds of screening and subculture; (c) determining one or more target genes for regulating the morphology and saponin accumulation of ginseng adventitious roots based on the candidate ginseng adventitious roots. This is also a method for determining target genes for regulating the morphology and saponin accumulation of ginseng adventitious roots.

[0027] Ginseng adventitious roots refer to the lateral roots growing from the rhizome of ginseng. Such roots are not directly or indirectly formed by the radicle and have no fixed growth position, so they are called adventitious roots. In some embodiments, ginseng adventitious roots can be formed by tissue culture technology. For example, ginseng adventitious roots can be formed by inducing ginseng callus and differentiation culture.

[0028] Ginsenoside is one of the main active ingredients of ginseng. It belongs to the triterpene glycosides and is formed by the condensation of sugar and aglycone.

[0029] Ethyl methanesulfonate (EMS) mutagenesis refers to the use of chemical mutagen ethyl methanesulfonate (EMS) to induce a high-density series of allele point mutations. EMS mutagenesis has the advantages of high efficiency, low negative effects, and easy operation.

[0030] Initial ginseng adventitious roots refer to healthy ginseng adventitious roots before mutagenesis treatment.

[0031] In some embodiments, mutagenesis treatment with EMS comprises the following steps.

[0032] S1: Take sterile ginseng adventitious root segments from initial ginseng adventitious roots.

[0033] In some embodiments, sterile ginseng adventitious root segments can be obtained in a variety of ways. In some embodiments, sterile ginseng adventitious root segments can be obtained in the following ways: washing the ginseng adventitious root segments, soaking and gently scrubbing the surface of the ginseng adventitious root segments with diluted soap solution, rinsing with tap water, sterilizing with 75% ethanol for 10-30 seconds, and then soaking in 4% sodium hypochlorite for 8-10 minutes.

[0034] S2: Prepare 3 / 4MS medium, add indole-3-butyric acid (IBA) and sucrose, sterilize, and cool for later use.

[0035] MS medium is a plant culture medium. 3 / 4MS medium is a medium in which the concentration of all elements in MS medium is reduced to 3 / 4 of the original, while retaining the basic components and proportions of MS medium.

[0036] In some embodiments, in step S2, the pH value of the 3 / 4MS culture medium is 5.5-6, the concentration of IBA is 2 mg / L-6 mg / L, and the amount of sucrose added is 1%-5% (mass ratio of sucrose to 3 / 4MS culture medium).

[0037] In some embodiments, the pH of the 3 / 4 MS medium may be 5.5, 5.6, 5.7, 5.8, 5.9, or 6. In some embodiments, the pH of the 3 / 4 MS medium may be 5.8.

[0038] In some embodiments, the concentration of IBA can be 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, or 6 mg / L. In some embodiments, the concentration of IBA can be 4 mg / L.

[0039] In some embodiments, the amount of sucrose added may be 1%, 2%, 3%, 4%, or 5%. In some embodiments, the amount of sucrose added may be 3%.

[0040] In some embodiments, in step S2, the pH value of the 3 / 4MS culture medium is 5.8, the concentration of IBA is 4 mg / L, and the amount of sucrose added is 3%.

[0041] In some embodiments, the 3 / 4MS medium can be sterilized by a variety of methods. In some embodiments, the 3 / 4MS medium can be sterilized by autoclaving at 121° C. for 20 minutes.

[0042] S3: Inoculate sterilized ginseng adventitious root segments into 3 / 4MS culture medium and culture at a constant temperature in the dark.

[0043] Constant temperature dark culture is a culture method carried out at a specific temperature without light.

[0044] In some embodiments, in step S3, the temperature of the constant temperature dark culture is 23°C-27°C.

[0045] In some embodiments, in step S3, the temperature of the constant temperature dark culture can be 23°C, 24°C, 26°C, or 27°C, etc.

[0046] In some embodiments, in step S3, the temperature of the constant temperature dark culture is 25°C.

[0047] In some embodiments, the EMS mutagenesis treatment of ginseng adventitious roots can be carried out by a variety of methods, such as immersion method, drip method, or co-cultivation method. In some embodiments, the EMS mutagenesis treatment of ginseng adventitious roots can be carried out by immersion method.

[0048] S4: Prepare EMS solution, soak the ginseng adventitious root segments after dark culture for 2 weeks in the EMS solution for EMS mutagenesis treatment, and rinse.

[0049] In some embodiments, in step S4, the concentration of the EMS solution is 0.1%-0.3%, and the soaking time is 50-70 minutes.

[0050] In some embodiments, in step S4, the concentration of the EMS solution is 0.2%, and the immersion time is 60 minutes.

[0051] In some embodiments, in step S4, the ginseng adventitious root segments are rinsed with sterile water to remove residual EMS solution.

[0052] S5: The adventitious roots of ginseng induced by EMS were cut into small pieces and inoculated into new 3 / 4MS medium and cultured for 4 weeks.

[0053] In some embodiments, the adventitious roots of ginseng treated with EMS were cut into small segments of 0.5 cm, inoculated into new 3 / 4 MS medium and cultured for 4 weeks.

[0054] Multiple rounds of screening refer to the process of repeated screening of ginseng adventitious roots. In some embodiments, multiple rounds of screening include primary screening and rescreening. Among them, primary screening refers to the first inoculation of ginseng adventitious root segments on a new 3 / 4MS medium after EMS mutagenesis treatment and culturing for 4 weeks, observing and recording the morphological changes of ginseng adventitious root segments, and selecting ginseng adventitious root segments with diverse morphologies and good growth; rescreening refers to observing and recording the morphological changes of ginseng adventitious root segments again after 4 weeks of subculture, and selecting ginseng adventitious root segments with stable and diverse morphologies.

[0055] Subculture, also known as "subculture" or "continuous culture", is a common operation in plant tissue culture used to maintain continuous production and proliferation of the culture.

[0056] In some embodiments, the subculture comprises inoculating the initially screened ginseng adventitious root segments into new 3 / 4MS culture medium again, and continuing to culture at a constant temperature of 25° C. in the dark for 4 weeks.

[0057] In some embodiments, subculturing comprises 15-25 consecutive subculturings.

[0058] In some embodiments, subculturing can include 15, 17, 19, 21, 23, or 25 consecutive subculturings.

[0059] In some embodiments, subculturing comprises 20 consecutive subculturings.

[0060] In some embodiments, the above subculture and screening process is repeated for a total of 20 rounds to obtain candidate ginseng adventitious roots with different morphologies and saponin accumulations.

[0061] The candidate ginseng adventitious roots refer to ginseng adventitious roots with different morphologies and saponin accumulations that are screened out through EMS mutagenesis, multiple rounds of screening, and subculture.

[0062] As used herein, "target gene" refers to one or more genes involved in regulating the morphology and saponin accumulation of ginseng adventitious roots.

[0063] In some embodiments, based on the candidate ginseng adventitious roots, determining one or more target genes that regulate the morphology and saponin accumulation of ginseng adventitious roots includes: extracting total RNA from the candidate ginseng adventitious roots and performing gene expression detection.

[0064] In some embodiments, the gene expression of the candidate ginseng adventitious roots can be detected using a variety of methods, such as real-time fluorescence quantitative analysis (qPCR), gene chip, or transcriptome sequencing.

[0065] In some embodiments, the gene expression detection is to detect the expression level of the target gene in the candidate ginseng adventitious roots using real-time fluorescence quantitative (qPCR) technology.

[0066] Quantitative Real-time PCR (qPCR) is a method that uses fluorescent chemicals to detect the total amount of products after each polymerase chain reaction (PCR) cycle in a DNA amplification reaction.

[0067] In some embodiments, the target genes include PgARF1, PgIAA02, PgCYCD3, PgEXPA1, PgWOX5, PgPIN3, PgLBD16, PgCYP450, PgSUT1, PgGASA4, PgHMGR1, PgFPS1, PgSS1, PgCYP716A47, PgUGT71A27, PgSE, PgDDS, PgCYP716A53v2, PgUGT74AE2, and Pgβ-AS.

[0068] Among them, PgARF1 is a key gene regulating the auxin signaling pathway, affecting adventitious root cell division and branching growth. PgIAA02 is a gene that inhibits auxin response and regulates root branching and cell differentiation. PgCYCD3 participates in cell cycle regulation and accelerates root cell division. PgEXPA1 is a gene that controls cell wall relaxation and affects root elongation growth. PgWOX5 is a gene that regulates root apical meristem and maintains root extension and branching. PgPIN3 is an auxin polar transport protein gene that regulates adventitious root branching and lateral root formation. PgLBD16 is a gene that promotes adventitious root development and participates in the cell differentiation process. PgCYP450 encodes cytochrome P450, participates in secondary metabolism, and affects root morphology and adaptability. PgSUT1 is a sucrose transporter gene that affects root carbohydrate supply. PgGASA4 regulates auxin signaling and cell proliferation and promotes root development. PgHMGR1 (3-hydroxy-3-methylglutaryl-CoA reductase) catalyzes the rate-limiting step of the mevalonate pathway and plays an important regulatory role in the formation of saponin precursors. PgFPS1 (geranyl pyrophosphate synthase) is involved in the early steps of the formation of terpenoids. PgSS1 (sabinene synthase) and PgSE (sabinene epoxidase) work together to produce sabinene, the precursor of saponin synthesis. PgCYP716A47 (cytochrome P450 monooxygenase) and PgCYP716A53v2 (cytochrome P450 enzyme) participate in specific oxidation steps and are responsible for further oxidative modification of saponins. PgUGT71A27 (UDP-glucosyltransferase) and PgUGT74AE2 (glycosyltransferase) are involved in the glycosylation modification of saponins. PgDDS (dammarene synthase) generates the dammarene ring structure, which is an important component of many ginsenosides. Pgβ-AS (β-amyrin synthase) produces protosapogenins, which plays an important role in the production of different types of saponins. By regulating the expression of specific genes, the length of ginseng adventitious roots, the number of branches, and the proliferation rate of cells can be affected, while also regulating the synthesis and accumulation of ginsenosides.

[0069] In some embodiments, a method for regulating the morphology and saponin accumulation of ginseng adventitious roots further comprises: regulating the morphology and saponin accumulation of the target ginseng adventitious roots by regulating the expression level of one or more target genes.

[0070] Target ginseng adventitious roots refer to ginseng adventitious roots with different morphologies and saponin accumulations obtained by regulating the expression levels of one or more target genes.

[0071] In some embodiments, by regulating the expression level of one or more target genes, precise regulation of the morphology and saponin accumulation of the target ginseng adventitious roots can be achieved. These target genes may include genes related to morphological development (such as PgARF1, PgIAA02, PgCYCD3, etc.) and genes related to saponin synthesis (such as PgHMGR1, PgFPS1, PgSS1, etc.). In some embodiments, the regulation of gene expression can be achieved by exogenous hormones, environmental conditions adjustment or gene editing technology, thereby optimizing the growth characteristics of ginseng adventitious roots and the accumulation of active ingredients.

[0072] In some embodiments, the saponin monomers in the selected ginseng adventitious roots are Rg1, Re, Rg2, Rg group, Rb1, Rc, Rb2, Rd, Rb group. In some embodiments, the saponin monomer content in the selected ginseng adventitious roots is 0.01%-5%, and the total saponin content is 1%-8%.

[0073] In some embodiments, the total saponin content in the candidate ginseng adventitious roots may be 2.0%-7.0%.

[0074] In some embodiments, the total saponin content in the candidate ginseng adventitious roots may be 2.0%, 6.0% or 7.0%.

[0075] In some embodiments, the content of Rg1 in adventitious roots of different forms is 0.018%-0.026%. In some embodiments, the content of Rg1 in adventitious roots of different forms can be 0.018%, 0.020%, 0.022%, 0.024% or 0.026%.

[0076] In some embodiments, the content of Re in adventitious roots of different forms is 0.1%-0.3%. In some embodiments, the content of Re in adventitious roots of different forms can be 0.1%, 0.2% or 0.3%.

[0077] In some embodiments, the content of Rg2 in adventitious roots of different forms is 0.01%-0.02%. In some embodiments, the content of Rg2 in adventitious roots of different forms can be 0.01%, 0.015% or 0.02%.

[0078] In some embodiments, the content of Rg group saponin in adventitious roots of different forms is 0.1%-0.35%. In some embodiments, the content of Rg group saponin in adventitious roots of different forms can be 0.1%, 0.2%, 0.3% or 0.35%.

[0079] In some embodiments, the content of Rb1 in adventitious roots of different shapes is 0.5%-1.8%. In some embodiments, the content of Rb1 in adventitious roots of different shapes can be 0.5%, 0.6%, 0.8%, 1.0%, 1.3%, 1.5% or 1.8%.

[0080] In some embodiments, the content of Rc in adventitious roots of different shapes is 0.4%-1.4%. In some embodiments, the content of Rc in adventitious roots of different shapes can be 0.4%, 0.6%, 0.8%, 1.0%, 1.2% or 1.4%.

[0081] In some embodiments, the content of Rb2 in adventitious roots of different shapes is 0.5%-1.3%. In some embodiments, the content of Rb2 in adventitious roots of different shapes can be 0.5%, 0.7%, 0.9%, 1.1%, or 1.3%.

[0082] In some embodiments, the content of Rd in adventitious roots of different shapes is 0.6%-1.5%. In some embodiments, the content of Rd in adventitious roots of different shapes can be 0.6%, 0.8%, 1.0%, 1.2% or 1.5%.

[0083] In some embodiments, the content of Rb group saponins in adventitious roots of different forms is 1.5%-4.0%.

[0084] In some embodiments, the content of Rb group saponins in adventitious roots of different forms is 1.5%, 1.8%, 2.0%, 2.2%, 2.5%, 3.0%, 3.5% or 4.0%.

[0085] The present specification also provides the application of target gene combination in regulating ginseng adventitious root morphology and saponin accumulation. In some embodiments, the target gene combination includes one or more of PgARF1, PgIAA02, PgCYCD3, PgEXPA1, PgWOX5, PgPIN3, PgLBD16, PgCYP450, PgSUT1, PgGASA4, PgHMGR1, PgFPS1, PgSS1, PgCYP716A47, PgUGT71A27, PgSE, PgDDS, PgCYP716A53v2, PgUGT74AE2 and Pgβ-AS.

[0086] In some embodiments, the target gene combination includes PgARF1, PgIAA02, PgCYCD3, PgEXPA1, PgWOX5, PgPIN3, PgLBD16, PgCYP450, PgSUT1, PgGASA4, PgHMGR1, PgFPS1, PgSS1, PgCYP716A47, PgUGT71A27, PgSE, PgDDS, PgCYP716A53v2, PgUGT74AE2 and Pgβ-AS.

[0087] In some embodiments, the target gene combination includes at least one of PgARF1, PgWOX5, PgLBD16, PgHMGR1, and PgSS1.

[0088] In some embodiments, optimization of ginseng adventitious root morphology is achieved by regulating the expression of one or more genes among PgARF1, PgIAA02, PgCYCD3, PgEXPA1, PgWOX5, PgPIN3, PgLBD16, PgCYP450, PgSUT1 and PgGASA4.

[0089] In some embodiments, the optimization of ginseng adventitious root morphology is achieved by regulating the expression of PgARF1, PgIAA02, PgCYCD3, PgEXPA1, PgWOX5, PgPIN3, PgLBD16, PgCYP450, PgSUT1 and PgGASA4 genes.

[0090] In some embodiments, the primer sequence for detecting the PgARF1 gene may have at least 95%, 97%, 98% or 99% similarity to any one of SEQ ID NO:3 and SEQ ID NO:4.

[0091] In some embodiments, the sequences of the primer pair for detecting the PgARF1 gene are shown in SEQ ID NO:3 and SEQ ID NO:4.

[0092] In some embodiments, the primer sequence for detecting the PgIAA02 gene may have at least 95%, 97%, 98% or 99% similarity to any one of SEQ ID NO:5 and SEQ ID NO:6.

[0093] In some embodiments, the sequences of the primer pair for detecting the PgIAA02 gene are shown in SEQ ID NO:5 and SEQ ID NO:6.

[0094] In some embodiments, the primer sequence for detecting the PgCYCD3 gene may have at least 95%, 97%, 98% or 99% similarity to any one of SEQ ID NO:7 and SEQ ID NO:8.

[0095] In some embodiments, the sequences of the primer pair for detecting the PgCYCD3 gene are shown in SEQ ID NO:7 and SEQ ID NO:8.

[0096] In some embodiments, the primer sequence for detecting the PgEXPA1 gene may have at least 95%, 97%, 98% or 99% similarity to any one of the sequences such as SEQ ID NO:9 and SEQ ID NO:10.

[0097] In some embodiments, the sequences of the primer pair for detecting the PgEXPA1 gene are shown in SEQ ID NO:9 and SEQ ID NO:10.

[0098] In some embodiments, the primer sequence for detecting the PgWOX5 gene may have at least 95%, 97%, 98% or 99% similarity to any one of the sequences of SEQ ID NO:11 and SEQ ID NO:12.

[0099] In some embodiments, the sequences of the primer pair for detecting the PgWOX5 gene are shown in SEQ ID NO:11 and SEQ ID NO:12.

[0100] In some embodiments, the primer sequence for detecting the PgPIN3 gene may have at least 95%, 97%, 98% or 99% similarity to any one of SEQ ID NO:13 and SEQ ID NO:14.

[0101] In some embodiments, the sequences of the primer pair for detecting the PgPIN3 gene are shown in SEQ ID NO:13 and SEQ ID NO:14.

[0102] In some embodiments, the primers for detecting the PgLBD16 gene may have at least 95%, 97%, 98% or 99% similarity to any one of the sequences of SEQ ID NO:15 and SEQ ID NO:16.

[0103] In some embodiments, the sequences of the primer pair for detecting the PgLBD16 gene are shown in SEQ ID NO:15 and SEQ ID NO:16.

[0104] In some embodiments, the primer sequence for detecting the PgCYP450 gene may have at least 95%, 97%, 98% or 99% similarity to any one of SEQ ID NO:17 and SEQ ID NO:18.

[0105] In some embodiments, the sequences of the primer pair for detecting the PgCYP450 gene are shown in SEQ ID NO:17 and SEQ ID NO:18.

[0106] In some embodiments, the primer sequence for detecting the PgSUT1 gene may have at least 95%, 97%, 98% or 99% similarity to any one of SEQ ID NO: 19 and SEQ ID NO: 20.

[0107] In some embodiments, the sequences of the primer pair for detecting the PgSUT1 gene are shown in SEQ ID NO: 19 and SEQ ID NO: 20.

[0108] In some embodiments, the primer sequence for detecting the PgGASA4 gene may have at least 95%, 97%, 98% or 99% similarity to any one of SEQ ID NO:21 and SEQ ID NO:22.

[0109] In some embodiments, the sequences of the primer pair for detecting the PgGASA4 gene are shown in SEQ ID NO:21 and SEQ ID NO:22.

[0110] In some embodiments, the synthesis and accumulation of ginseng adventitious root saponins are promoted by regulating the expression of one or more genes among PgHMGR1, PgFPS1, PgSS1, PgCYP716A47, PgUGT71A27, PgSE, PgDDS, PgCYP716A53v2, PgUGT74AE2, and Pgβ-AS.

[0111] In some embodiments, the synthesis and accumulation of ginseng adventitious root saponins are promoted by regulating the expression of PgHMGR1, PgFPS1, PgSS1, PgCYP716A47, PgUGT71A27, PgSE, PgDDS, PgCYP716A53v2, PgUGT74AE2, and Pgβ-AS genes.

[0112] In some embodiments, the primer sequence for detecting the PgHMGR1 gene may have at least 95%, 97%, 98% or 99% similarity to any one of SEQ ID NO:23 and SEQ ID NO:24.

[0113] In some embodiments, the sequences of the primer pair for detecting the PgHMGR1 gene are shown in SEQ ID NO:23 and SEQ ID NO:24.

[0114] In some embodiments, the primer sequence for detecting the PgFPS1 gene may have at least 95%, 97%, 98% or 99% similarity to any one of SEQ ID NO:25 and SEQ ID NO:26.

[0115] In some embodiments, the sequences of the primer pair for detecting the PgFPS1 gene are shown in SEQ ID NO:25 and SEQ ID NO:26.

[0116] In some embodiments, the primers for detecting the PgSS1 gene may have at least 95%, 97%, 98% or 99% similarity to any one of the sequences of SEQ ID NO:27 and SEQ ID NO:28.

[0117] In some embodiments, the sequences of the primer pair for detecting the PgSS1 gene are shown in SEQ ID NO:27 and SEQ ID NO:28.

[0118] In some embodiments, the primer sequence for detecting the PgCYP716A47 gene may have at least 95%, 97%, 98% or 99% similarity to any one of SEQ ID NO:29 and SEQ ID NO:30.

[0119] In some embodiments, the sequences of the primer pair for detecting the PgCYP716A47 gene are shown in SEQ ID NO:29 and SEQ ID NO:30.

[0120] In some embodiments, the primer sequence for detecting the PgUGT71A27 gene may have at least 95%, 97%, 98% or 99% similarity to any one of the sequences SEQ ID NO:31 and SEQ ID NO:32.

[0121] In some embodiments, the sequences of the primer pair for detecting the PgUGT71A27 gene are shown in SEQ ID NO:31 and SEQ ID NO:32.

[0122] In some embodiments, the primer sequence for detecting the PgSE gene may have at least 95%, 97%, 98% or 99% similarity to any one of SEQ ID NO:33 and SEQ ID NO:34.

[0123] In some embodiments, the sequences of the primer pair for detecting the PgSE gene are shown in SEQ ID NO:33 and SEQ ID NO:34.

[0124] In some embodiments, the primer sequence for detecting the PgDDS gene may have at least 95%, 97%, 98% or 99% similarity to any one of SEQ ID NO:35 and SEQ ID NO:36.

[0125] In some embodiments, the sequences of the primer pair for detecting the PgDDS gene are shown in SEQ ID NO:35 and SEQ ID NO:36.

[0126] In some embodiments, the primer sequence for detecting the PgCYP716A53v2 gene may have at least 95%, 97%, 98% or 99% similarity to any one of SEQ ID NO:37 and SEQ ID NO:38.

[0127] In some embodiments, the sequences of the primer pair for detecting the PgCYP716A53v2 gene are shown in SEQ ID NO:37 and SEQ ID NO:38.

[0128] In some embodiments, the primer sequence for detecting the PgUGT74AE2 gene may have at least 95%, 97%, 98% or 99% similarity to any one of SEQ ID NO:39 and SEQ ID NO:40.

[0129] In some embodiments, the sequences of the primer pair for detecting the PgUGT74AE2 gene are shown in SEQ ID NO:39 and SEQ ID NO:40.

[0130] In some embodiments, the primer sequence for detecting the Pgβ-AS gene may have at least 95%, 97%, 98% or 99% similarity to any one of SEQ ID NO:41 and SEQ ID NO:42.

[0131] In some embodiments, the sequences of the primer pair for detecting the Pgβ-AS gene are shown in SEQ ID NO:41 and SEQ ID NO:42.

[0132] The examples of this specification adopt the EMS mutagenesis method to treat ginseng adventitious roots through chemical mutagenesis to produce morphological variations. After mutagenesis treatment, morphologically stable and diverse ginseng adventitious roots were obtained through multiple rounds of screening and subculture. Five different morphologies of ginseng adventitious roots were obtained in the examples of this specification. By regulating the expression and metabolic pathways of key genes, the cell division and differentiation process of ginseng adventitious roots are controlled, and ultimately different root morphologies are formed.

[0133] The embodiments of this specification lay a solid technical foundation for the efficient cultivation and ingredient development of ginseng by precisely controlling the key factors that affect the morphology of adventitious roots. The embodiments of this specification achieve effective regulation of the morphology of ginseng adventitious roots and the synthesis of its secondary metabolites (such as ginsenosides), promote the accumulation of ginsenosides, thereby enhancing the medicinal value of ginseng, meeting the market demand for high-quality ginseng products, and providing reliable technical support for the industrial production of medicinal plants, which has significant innovation, practicality and commercial value. Example Example 1: Screening of ginseng adventitious root morphology

[0134] 1. Material preparation: Take sterile adventitious root segments of healthy ginseng plants, about 0.5 cm in length; prepare 3 / 4MS culture medium, adjust the pH to 5.8, add 4 mg / L IBA and 3% sucrose; sterilize the culture medium at 121°C for 20 minutes, and cool it for later use.

[0135] 2. Inoculate the adventitious root segments from healthy ginseng plants into the prepared 3 / 4MS culture medium. After inoculation, place the culture dish in a constant temperature environment of 25℃ and maintain dark culture conditions.

[0136] 3. Prepare 0.2% ethyl methanesulfonic acid (EMS) solution and soak the adventitious root segments after 2 weeks of culture in the EMS solution for 60 minutes. After the treatment, rinse with sterile water to remove EMS residues.

[0137] 4. After EMS mutagenesis treatment, the ginseng adventitious roots were cut into small segments of 0.5 cm to obtain at least 20,000 small segments. After growing on the new culture medium for 4 weeks, the morphological changes of the adventitious roots were observed.

[0138] 5. Verification of the stability of morphological traits of ginseng adventitious roots: After removing the ginseng adventitious root segments that were morphologically identical to RS-A, four ginseng adventitious roots (RS-B, RS-C, RS-D, and RS-E) that were different in morphology from RS-A were finally screened out after 20 consecutive subcultures.

[0139] 6. The morphological characteristics of ginseng adventitious roots are shown in Figure 1As shown in Table 1, RS-A (control): lateral roots are closely arranged and dense, with a short root cluster structure; RS-B: the main root is slender and has few branches; RS-C: the root is long and has obvious branches, about 5-6 cm in length, with a moderate diameter; RS-D: the root is relatively strong and has the largest number of branches; RS-E: the main root is thin and has very few branches. These different forms of ginseng adventitious roots provide a rich material basis for subsequent studies on gene expression regulation and saponin accumulation. Table 1 Example 2: Changes in expression levels of key genes for root morphological development

[0140] This example aims to study the expression changes of 10 key genes related to the morphological development of ginseng adventitious roots under different process conditions. These genes are involved in auxin signal transduction, cell cycle regulation, cell wall synthesis, and secondary metabolic pathways, and may have an important impact on the morphological characteristics of ginseng adventitious roots, such as length, number of branches, and cell proliferation rate.

[0141] 1. Five different forms of ginseng adventitious roots (RS-A, RS-B, RS-C, RS-D and RS-E) grown in culture medium (3 / 4MS culture medium, pH adjusted to 5.8, added with 4 mg / L IBA and 3% sucrose) for 30 days were selected.

[0142] 2. Gene selection: Ten key genes related to adventitious root morphology and development were selected, including PgARF1, PgIAA02, PgCYCD3, PgEXPA1, PgWOX5, PgPIN3, PgLBD16, PgCYP450, PgSUT1 and PgGASA4. The functions of these genes are summarized as follows: PgARF1: a key gene regulating the auxin signaling pathway, affecting adventitious root cell division and branching growth; PgIAA02: a gene that inhibits auxin response and regulates root branching and cell differentiation; PgCYCD3: involved in cell cycle regulation, accelerating root cell division; PgEXPA1: a gene that controls cell wall relaxation and affects root elongation growth; PgWOX5: a gene that regulates root apical meristem and maintains root extension and branching; PgPIN3: an auxin polarity transport protein gene that regulates adventitious root branching and lateral root formation; PgLBD16: a gene that promotes adventitious root development and participates in the cell differentiation process; PgCYP450: encoding cytochrome P450, involved in secondary metabolism, affecting root morphology and adaptability; PgSUT1: a sucrose transporter gene that affects root carbohydrate supply; PgGASA4: regulates auxin signaling and cell proliferation and promotes root development.

[0143] 3. RNA extraction and reverse transcription: Use TRIzol reagent to extract total RNA from ginseng adventitious roots according to the kit instructions, and use a reverse transcription kit to reverse transcribe RNA into cDNA.

[0144] 4. Use the SYBR Green qPCR kit to perform qPCR detection according to standard procedures, use Actin as the internal reference gene, and calculate the relative expression of the target gene. Table 2 shows the key genes related to adventitious root morphology and their corresponding detection primers. Table 2

[0145] 5. The results are as follows Figure 2 As shown in the figure, by comparing the expression differences of key genes in the above five morphological adventitious roots, the following patterns can be observed: taking the expression of related genes in RS-A as a reference, RS-B type adventitious roots promoted lateral root growth, among which the expression of PgPIN3 and PgCYCD3 increased, indicating an increase in the number of branches; in RS-C type adventitious roots, the expression of cell cycle and cell wall related genes (such as PgEXPA1, PgCYCD3) was at a medium level, which promoted the moderate extension growth of adventitious roots; in RS-D type adventitious roots, the expression of genes such as PgWOX5, PgLBD16 and PgCYP450 was high, showing a longer root system and stronger branching growth; RS-E type adventitious roots showed the highest gene expression level, among which PgCYP450, PgWOX5 and PgLBD16 were significantly expressed under this condition, resulting in the maximum number of root branches and growth rate, which makes RS-E type adventitious roots particularly suitable for meeting the cultivation needs of high yields.

[0146] Relative to RS-A, the expression levels of PgPIN3 and PgCYCD3 in RS-B were 3.7-fold and 2.6-fold, respectively, and the expression levels of other genes were as follows: PgARF1 1.5-fold, PgIAA02 1.4-fold, PgEXPA1 2.3-fold, PgWOX5 1.2-fold, PgLBD16 1.6-fold, PgCYP450 1.4-fold, PgSUT1 1.5-fold, and PgGASA4 3.3-fold;

[0147] RS-C: The expression of cell cycle and cell wall related genes (such as PgEXPA1 and PgCYCD3) was moderate, with the expression levels of PgEXPA1 and PgCYCD3 being 1.5-fold and 2.4-fold, respectively. The expression levels of other genes were as follows: PgARF1 was 1.2-fold, PgIAA02 was 3.3-fold, PgWOX5 was 4.1-fold, PgPIN3 was 1.3-fold, PgLBD16 was 1.2-fold, PgCYP450 was 3.2-fold, PgSUT1 was 5.4-fold, and PgGASA4 was 1.2-fold;

[0148] RS-D: The expression of PgWOX5, PgLBD16 and PgCYP450 genes was high, with the expression levels of PgWOX5, PgLBD16 and PgCYP450 being 2-fold, 1.3-fold and 2.5-fold, respectively. The expression levels of other genes were as follows: PgARF1 was 4.1-fold, PgIAA02 was 2.1-fold, PgCYCD3 was 1.9-fold, PgEXPA1 was 2.2-fold, PgPIN3 was 1.8-fold, PgSUT1 was 1.8-fold, and PgGASA4 was 2.2-fold;

[0149] RS-E: The expression of multiple genes (such as PgCYP450, PgWOX5, PgLBD16, etc.) was significantly increased. The expression levels of PgCYP450, PgWOX5 and PgLBD16 were 2.9 times, 2.8 times and 3.7 times, respectively. The expression levels of other genes are as follows: PgARF1 was 3.5 times, PgIAA02 was 2.3 times, PgCYCD3 was 2.4 times, PgEXPA1 was 2.6 times, PgPIN3 was 2.5 times, PgSUT1 was 2.4 times, and PgGASA4 was 1.6 times. Example 3: Expression analysis of key enzyme genes for ginsenoside synthesis

[0150] The present example aims to analyze the changes in the expression levels of key enzyme genes related to ginsenoside synthesis in RS-A, RS-B, RS-C, RS-D and RS-E ginseng adventitious roots.

[0151] The ginseng adventitious root material in Example 2 was used as a sample, and 10 key enzyme genes closely related to the ginsenoside biosynthesis pathway were selected. The expression differences of these genes in adventitious roots of different morphologies were determined by quantitative polymerase chain reaction (qPCR) technology. The 10 selected key genes and their functions in the saponin synthesis pathway are as follows: PgHMGR1 (3-hydroxy-3-methylglutaryl-CoA reductase) catalyzes the rate-limiting step of the mevalonate pathway; PgFPS1 (geranyl pyrophosphate synthase) is involved in the early steps of the production of terpenoids; PgSS1 (sabinene synthase) catalyzes the production of sabinene, a precursor substance for saponin synthesis; PgCYP716A47 (cytochrome P450 monooxygenase) is involved in the conversion of sabinene to Oxidation reaction of sapogenin; PgUGT71A27 (UDP-glucosyltransferase) participates in the glycosylation reaction of sapogenin; PgSE (sabinene epoxidase) regulates the further cyclization of sabinene; PgDDS (dammarene synthase) is the key enzyme for the production of dammarene; PgCYP716A53v2 (cytochrome P450 enzyme) involves multi-step oxidation reactions in secondary metabolic pathways; PgUGT74AE2 (glycosyltransferase) participates in the terminal glycosylation of saponins. Pgβ-AS (β-amyrin synthase) plays an important role in the generation of saponin synthesis precursors. Table 3 shows the key genes related to ginsenoside synthesis and their corresponding detection primers. Table 3

[0152] The results are as follows Figure 3 As shown in the figure, the expression levels of each key gene in the ginsenoside synthesis pathway showed volatility under different process conditions. Specifically, the expression level of PgCYP716A47 reached the maximum in RS-C, with an increase of 9.9 times, while in RS-B and RS-E, it was 2.5 times and 3.4 times, respectively, showing a lower expression level. In addition, PgDDS and PgCYP716A53v2 reached high expression levels of 10 times and 9.8 times in RS-E, respectively, while showing obvious low volatility in other adventitious roots. Overall, the expression levels of multiple key enzymes in RS-E type adventitious roots reached peak values, indicating that the RS-E process conditions may be most conducive to increasing the total yield of ginsenosides.

[0153] PgHMGR1 is a key enzyme in the mevalonate pathway. In the early stage of saponin biosynthesis, PgHMGR1 (3-hydroxy-3-methylglutaryl-CoA reductase) plays an important regulatory role in the production of saponin precursors by catalyzing the rate-limiting step of the mevalonate (MVA) pathway. Figure 3As shown, the expression of PgHMGR1 was significantly upregulated in RS-D (9.1-fold), and the expression was lower in RS-B and RS-C (2.3-3.8-fold), which may indicate that the initial stage of the MVA pathway was significantly activated in RS-D, laying the foundation for the subsequent accumulation of saponin precursor substances.

[0154] PgFPS1 (geranyl pyrophosphate synthase) is a synthase of geranyl pyrophosphate (FPP), responsible for the production of terpenoid precursors of saponins. The expression of PgFPS1 was higher in RS-B (5.4-fold) and RS-D (7.6-fold), while it decreased to 2.9-fold in RS-E, indicating that the enzyme may be more sensitive to terpenoid synthesis under specific conditions. The high expression in RS-D may be related to the increase in FPP synthesis, which is conducive to the accumulation of saponin precursors.

[0155] PgSS1 (sabinene synthase) and PgSE (sabinene epoxidase) work together to produce sabinene, a precursor for saponin synthesis. PgSS1 is highly expressed in RS-D and RS-E, especially in RS-E, where the expression level reaches 9.1 times, indicating that RS-E may be conducive to sabinene accumulation and provide more precursors for downstream saponin synthesis. PgSE also showed the highest expression level (9.6 times) in RS-D, suggesting that adventitious roots of RS-D and RS-E morphologies may promote the production of saponin precursors by increasing the expression levels of sabinene-related enzymes.

[0156] PgCYP716A47 and PgCYP716A53v2 belong to the cytochrome P450 (CYP450) enzyme family and are responsible for specific oxidation steps in saponin biosynthesis. The expression level of PgCYP716A47 reached the highest level in RS-C (9.9 times), while it fluctuated greatly in other morphological roots, suggesting that the expression of this gene was significantly stimulated by conditions. The expression level of PgCYP716A53v2 also reached a high level in RS-E (9.8 times), indicating that RS-E may enhance the activity of P450 enzymes, which is conducive to further oxidative modification of saponins and the production of different saponin types.

[0157] PgUGT71A27 and PgUGT74AE2, as UDP-glucosyltransferases, regulate the glycosylation modification of saponins under different conditions. PgUGT71A27 was expressed at the highest level in RS-E (8.3 times), indicating that in the adventitious roots of RS-E, PgUGT71A27 may be beneficial to the glycosylation modification of the final saponin product, making it more biologically active or easier to accumulate. PgUGT74AE2 was expressed at the highest level in RS-B (7.1 times), which may be related to the specific saponin metabolism requirements under this condition.

[0158] PgDDS is responsible for the generation of the dammarene ring structure, which is an important component of many ginsenosides. PgDDS was expressed at the highest level (10 times) under RS-E conditions, and also showed high expression levels in RS-B and RS-D, indicating that RS-E and RS-D may be conducive to the accumulation of dammarene and have a significant impact on the total yield of ginsenosides.

[0159] Pgβ-AS is the synthase of β-amyrin, a saponin precursor, and plays a vital role in the production of protosapogenin. The expression levels of Pgβ-AS were higher in RS-C and RS-E, 8.1 and 7.2 times, respectively, indicating that these two conditions are conducive to improving the synthesis of protosapogenin, thus providing a raw material basis for the diversification of saponins. Example 4: Induction of ginsenosides from ginseng adventitious roots of different forms

[0160] 1. Take sterile adventitious root segments of healthy ginseng plants, about 0.5 cm in length; prepare 3 / 4MS culture medium, adjust the pH to 5.8, add 4 mg / L IBA and 3% sucrose, sterilize the culture medium at 121°C for 20 minutes, and cool for use.

[0161] 2. Inoculate the adventitious root segments of ginseng of five morphologies (RS-A, RS-B, RS-C, RS-D and RS-E) into the prepared 3 / 4MS liquid culture medium and culture them in the dark at a constant temperature of 25°C.

[0162] 3. After 4 weeks of culture, add methyl jasmonate (MeJA) to the culture medium at a final concentration of 100 μM, and continue to induce culture at 25°C in the dark for 2 weeks. MeJA is an important plant hormone that can significantly increase the yield of various secondary metabolites in ginseng.

[0163] 4. After the induction culture, the collected samples were dried at 55°C until constant weight, and the saponin monomer and total saponin content were detected by high performance liquid chromatography (HPLC).

[0164] Figure 4 The content of various saponins in different forms of ginseng adventitious roots after cultivation is different according to some examples of this specification. Figure 4 A): The content of Rg1 in RS-A (T1) is the lowest, about 0.018%. The content of Rg1 increases from RS-B to RS-E, and is close to 0.026% in RS-D (T4) and RS-E (T5), which is the highest value, indicating that the biosynthesis pathway of Rg1 in RS-D and RS-E has been strengthened. Re content ( Figure 4B): The content of Re in RS-A (T1) is 0.1%, which significantly increases to about 0.3% in RS-C (T3) to RS-E (T5), indicating that the synthesis of Re is more active in these two forms of adventitious roots. Rg2 content ( Figure 4 C): The content of Rg2 was the lowest in RS-A (about 0.01%), while it increased in RS-B (T2) and RS-D (T4) to about 0.02%, indicating that the biosynthesis of Rg2 in RS-B and RS-D was enhanced. Figure 4 D): The content of Rg group saponins was the lowest in RS-A, while it showed a higher accumulation in RS-B, RS-D, and RS-E. In particular, the Rg content reached the highest in RS-E, which was 0.35%. Figure 4 E): The content of Rb1 in RS-A is low (about 0.5%), but it is significantly increased to 1.8% in RS-C and RS-E. This result shows that the biosynthesis of Rb1 in RS-C and RS-E is significantly enhanced. Figure 4 F): In RS-A, the Rc content is about 0.4%, while in RS-C and RS-E it increases significantly to more than 1.4%, indicating that these conditions promote the synthesis of Rc. Rb2 content ( Figure 4 The content of Rb2 was the lowest in RS-A (about 0.5%), and significantly increased to 1.2% and 1.3% in RS-D and RS-E, indicating that the biosynthesis pathway of Rb2 was significantly activated in these two forms of adventitious roots. Figure 4 H in the Rd content: Rd was the lowest in RS-A (about 0.6%), and the highest in RS-B and RS-E, close to 1.5%, indicating that the biosynthesis of Rd in the adventitious roots of RS-B and RS-E was significantly enhanced. Rb content ( Figure 4 I): The Rb content is low in RS-A (about 1.5%), but increases significantly to about 4% in RS-B, RS-D and RS-E. Rb / Rg ratio ( Figure 4 The Rb / Rg ratios in RS-B, RS-D, and RS-E were relatively high, especially in RS-E, which indicated that the biosynthesis of Rb saponins was more active in RS-E. Figure 4 The total saponin content was the lowest under RS-A, about 2%, while it reached 6% and 7% in RS-D and RS-E, respectively. Figure 4 L): The total saponin content in RS-A was about 200 mg / L, while it increased to 800 mg / L and 900 mg / L in RS-D and RS-E, respectively, which were significantly higher than those in other conditions.

[0165] By adding MeJA to induce different forms of ginseng adventitious roots, it was found that RS-D and RS-E adventitious roots showed significant increases in the content of multiple saponin monomers and total saponins. This indicates that these forms of adventitious roots have higher potential in saponin synthesis, providing strong support for subsequent industrial production and improvement of medicinal value.

[0166] The basic concepts have been described above. Obviously, for those skilled in the art, the above detailed disclosure is only for example and does not constitute a limitation of this specification. Although not explicitly stated here, those skilled in the art may make various modifications, improvements and corrections to this specification. Such modifications, improvements and corrections are suggested in this specification, so such modifications, improvements and corrections still belong to the spirit and scope of the exemplary embodiments of this specification.

[0167] At the same time, this specification uses specific words to describe the embodiments of this specification. For example, "one embodiment", "an embodiment", and / or "some embodiments" refer to a certain feature, structure or characteristic related to at least one embodiment of this specification. Therefore, it should be emphasized and noted that "one embodiment" or "an embodiment" or "an alternative embodiment" mentioned twice or more in different positions in this specification does not necessarily refer to the same embodiment. In addition, certain features, structures or characteristics in one or more embodiments of this specification can be appropriately combined.

[0168] In addition, unless explicitly stated in the claims, the order of the processing elements and sequences described in this specification, the use of alphanumeric characters, or the use of other names are not intended to limit the order of the processes and methods of this specification. Although the above disclosure discusses some invention embodiments that are currently considered useful through various examples, it should be understood that such details are only for illustrative purposes, and the attached claims are not limited to the disclosed embodiments. On the contrary, the claims are intended to cover all modifications and equivalent combinations that are consistent with the essence and scope of the embodiments of this specification.

[0169] Similarly, it should be noted that in order to simplify the description disclosed in this specification and thus help understand one or more embodiments of the invention, in the above description of the embodiments of this specification, multiple features are sometimes combined into one embodiment, figure or description thereof. However, this disclosure method does not mean that the features required by the subject matter of this specification are more than the features mentioned in the claims. In fact, the features of the embodiments are less than all the features of the single embodiment disclosed above.

[0170] In some embodiments, numbers describing the number of components and attributes are used. It should be understood that such numbers used in the description of the embodiments are modified by the modifiers "about", "approximately" or "substantially" in some examples. Unless otherwise specified, "about", "approximately" or "substantially" indicate that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may change according to the required features of individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and adopt the general method of retaining digits. Although the numerical domains and parameters used to confirm the breadth of their range in some embodiments of this specification are approximate values, in specific embodiments, the setting of such numerical values ​​is as accurate as possible within the feasible range.

[0171] Each patent, patent application, patent application publication, and other materials, such as articles, books, specifications, publications, documents, etc., cited in this specification are hereby incorporated by reference in their entirety. Except for application history documents that are inconsistent with or conflicting with the contents of this specification, documents that limit the broadest scope of the claims of this specification (currently or later attached to this specification) are also excluded. It should be noted that if the descriptions, definitions, and / or use of terms in the materials attached to this specification are inconsistent or conflicting with the contents described in this specification, the descriptions, definitions, and / or use of terms in this specification shall prevail.

[0172] Finally, it should be understood that the embodiments described in this specification are only used to illustrate the principles of the embodiments of this specification. Other variations may also fall within the scope of this specification. Therefore, as an example and not a limitation, alternative configurations of the embodiments of this specification may be considered consistent with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments explicitly introduced and described in this specification.

Claims

1. A method for regulating the morphology and saponin accumulation of ginseng adventitious roots, characterized in that: The method comprises: (a) Initial ginseng adventitious roots were mutagenized with ethyl methanesulfonate (EMS); (b) After multiple rounds of screening and subculture, candidate ginseng adventitious roots with different morphologies and saponin accumulations are obtained; (c) determining one or more target genes that regulate the morphology and saponin accumulation of ginseng adventitious roots based on the candidate ginseng adventitious roots.

2. The method for regulating ginseng adventitious root morphology and saponin accumulation according to claim 1, characterized in that: The subculture comprises 15-25 consecutive subcultures.

3. The method for regulating ginseng adventitious root morphology and saponin accumulation according to claim 1, characterized in that: The EMS mutagenesis treatment The following steps are involved: S1: taking a sterilized ginseng adventitious root segment from the initial ginseng adventitious root; S2: Prepare 3 / 4MS medium, add indole-3-butyric acid (IBA) and sucrose, sterilize, and cool for later use; S3: inoculating the sterilized ginseng adventitious root segment into the 3 / 4MS medium and culturing in the dark at a constant temperature; S4: preparing an EMS solution, soaking the ginseng adventitious root segments after dark culture for 2 weeks in the EMS solution for the EMS mutagenesis treatment, and then rinsing; S5: Cut the ginseng adventitious roots after EMS mutagenesis into small pieces, inoculate them on new 3 / 4 MS medium and culture them for 4 weeks.

4. The method for regulating ginseng adventitious root morphology and saponin accumulation according to claim 3, characterized in that: In step S2, the pH value of the 3 / 4MS culture medium is 5.5-6, the concentration of the IBA is 2 mg / L-6 mg / L, and the amount of sucrose added is 1%-5%.

5. The method for regulating ginseng adventitious root morphology and saponin accumulation according to claim 3, characterized in that: In step S3, the temperature of the constant temperature dark culture is 23°C-27°C.

6. The method for regulating ginseng adventitious root morphology and saponin accumulation according to claim 3, characterized in that: In step S4, the concentration of the EMS solution is 0.1%-0.3%, and the soaking time is 50-70 minutes.

7. The method for regulating ginseng adventitious root morphology and saponin accumulation according to claim 1, characterized in that: The step of determining one or more target genes that regulate the morphology and saponin accumulation of ginseng adventitious roots based on the candidate ginseng adventitious roots comprises: The total RNA of the candidate ginseng adventitious roots is extracted to perform gene expression detection.

8. The method for regulating ginseng adventitious root morphology and saponin accumulation according to claim 7, characterized in that: The gene expression detection is to detect the expression amount of the target gene in the adventitious roots of the candidate ginseng using real-time fluorescence quantitative qPCR technology.

9. The method for regulating ginseng adventitious root morphology and saponin accumulation according to claim 1, characterized in that: The target genes include PgARF1, PgIAA02, PgCYCD3, PgEXPA1, PgWOX5, PgPIN3, PgLBD16, PgCYP450, PgSUT1, PgGASA4, PgHMGR1, PgFPS1, PgSS1, PgCYP716A47, PgUGT71A27, PgSE, PgDDS, PgCYP716A53v2, PgUGT74AE2 and Pgβ-AS.

10. The method for regulating ginseng adventitious root morphology and saponin accumulation according to claim 1, characterized in that: The method further comprises: regulating the morphology and saponin accumulation of the target ginseng adventitious roots by regulating the expression level of the one or more target genes.

11. The method for regulating ginseng adventitious root morphology and saponin accumulation according to claim 10, characterized in that: The expression level of the one or more target genes is regulated by exogenous hormones, environmental condition adjustment or gene editing technology.

12. The method for regulating ginseng adventitious root morphology and saponin accumulation according to claim 1, characterized in that: The content of saponin monomers in the selected ginseng adventitious roots is 0.01%-5%, and the content of total saponins is 1%-8%; the saponin monomers are Rg1, Re, Rg2, Rg group, Rb1, Rc, Rb2, Rd, and Rb group.

13. Application of target gene combination in regulating morphology and saponin accumulation of ginseng adventitious roots, characterized in that: The target gene combination includes one or more of PgARF1, PgIAA02, PgCYCD3, PgEXPA1, PgWOX5, PgPIN3, PgLBD16, PgCYP450, PgSUT1, PgGASA4, PgHMGR1, PgFPS1, PgSS1, PgCYP716A47, PgUGT71A27, PgSE, PgDDS, PgCYP716A53v2, PgUGT74AE2 and Pgβ-AS.

14. The use of the target gene combination according to claim 13 in regulating the morphology and saponin accumulation of ginseng adventitious roots, characterized in that: The target gene combination includes PgARF1, PgIAA02, PgCYCD3, PgEXPA1, PgWOX5, PgPIN3, PgLBD16, PgCYP450, PgSUT1, PgGASA4, PgHMGR1, PgFPS1, PgSS1, PgCYP716A47, PgUGT71A27, PgSE, PgDDS, PgCYP716A53v2, PgUGT74AE2 and Pgβ-AS.

15. The use of the target gene combination according to claim 13 in regulating the morphology and saponin accumulation of ginseng adventitious roots, characterized in that: The target gene combination includes at least one of PgARF1, PgWOX5, PgLBD16 PgHMGR1 and PgSS1.

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